In Search of Small Molecules That Selectively Inhibit MBOAT4

Ghrelin is a 28-residue peptide hormone produced by stomach P/D1 cells located in oxyntic glands of the fundus mucosa. Post-translational octanoylation of its Ser-3 residue, catalyzed by MBOAT4 (aka ghrelin O-acyl transferase (GOAT)), is essential for the binding of the hormone to its receptor in target tissues. Physiological roles of acyl ghrelin include the regulation of food intake, growth hormone secretion from the pituitary, and inhibition of insulin secretion from the pancreas. Here, we describe a medicinal chemistry campaign that led to the identification of small lipopeptidomimetics that inhibit GOAT in vitro. These molecules compete directly for substrate binding. We further describe the synthesis of heterocyclic inhibitors that compete at the acyl coenzyme A binding site.

Peroxiredoxin-6 is a Guardian of lung pathophysiologies

: The moonlighting protein, Prdx6 exhibits peroxidase activity, phospholipase activity and lysophosphatidylcholine acyl transferase (LPCAT) activity. Although it is ubiquitous in expression, its level is prominently high in the lung. Prdx6 has been known to be an important enzyme for the maintenance of normal lung physiologies including, anti-oxidant defense, lung surfactant homeostasis and cell signaling. Studies further unveiled that the altered activity (peroxidase or aiPLA2) of this enzyme is linked with various lung pathologies or diseases. In the present article, we attempted to address the various pathophysiologies or disease conditions (like lung ischemia, hyperoxia, lung cancer, emphysema and acute lung injury) wherein prdx6 is involved. The study implicates that Prdx6 could be used as a common drug target for multiple lung diseases. Important future insights have also been incorporated.

Determination of the membrane topology of PORCN, an O-acyl transferase that modifies Wnt signalling proteins

Wnt gradients elicit distinct cellular responses, such as proliferation, specification, differentiation and survival in a dose-dependent manner. Porcupine (PORCN), a membrane-bound O-acyl transferase (MBOAT) that resides in the endoplasmic reticulum, catalyses the addition of monounsaturated palmitate to Wnt proteins and is required for Wnt gradient formation and signalling. In humans, PORCN mutations are causal for focal dermal hypoplasia (FDH), an X-linked dominant syndrome characterized by defects in mesodermal and endodermal tissues. PORCN is also an emerging target for cancer therapeutics. Despite the importance of this enzyme, its structure remains poorly understood. Recently, the crystal structure of DltB, an MBOAT family member from bacteria, was solved. In this report, we use experimental data along with homology modelling to DltB to determine the membrane topology of PORCN. Our studies reveal that PORCN has 11 membrane domains, comprising nine transmembrane spanning domains and two reentrant domains. The N-terminus is oriented towards the lumen while the C-terminus is oriented towards the cytosol. Like DltB, PORCN has a funnel-like structure that is encapsulated by multiple membrane-spanning helices. This new model for PORCN topology allows us to map residues that are important for biological activity (and implicated in FDH) onto its three-dimensional structure.

A novel route for aspartame production by combining enzymatic and chemical reactions for industrial use

ABSTRACT Here, we report a novel industrial aspartame production route, involving the enzymatic production of α-l-aspartyl-l-phenylalanine β-methylester from l-aspartic acid dimethylester and l-phenylalanine by α-amino acid ester acyl transferase. The route also involves the chemical transformation of α-l-aspartyl-l-phenylalanine β-methylester to α-l-aspartyl-l-phenylalanine methylester hydrochloride (aspartame hydrochloride) in an aqueous solution with methanol and HCl, followed by HCl removal to form aspartame.

Palmitoylation of KChIP3 controls baseline mucin secretion

Baseline mucin secretion (BMS) is independent of external agonists and controlled by a small calcium binding protein named KChIP3. KChIP3 hosting mucin granules are not released until intracellular cytosolic calcium oscillations reach a threshold, KChIP3 binds calcium and detaches from granules, allowing their fusion to plasma membrane. Loss of KChIP3 or blocking its membrane attachment causes mucin hypersecretion. How is KChIP3 recruited to mucin granules? We show here that zDHHC (aspartate-histidine-histidine-cysteine motif in a cysteine-rich, zinc finger like domain) S-acyl-transferase dependent palmitoylation modulates binding of KChIP3 to mucin granules thereby affecting mucin secretion. We have found that inhibiting zDHHC-mediated palmitoylation in differentiated HT29-18N2, which express the Golgi-localized zDHHC3 and zDHHC4, releases KChIP3 from mucin granules and increases baseline mucin secretion. Mutation of the palmitoylation sites in KChIP3 (Cysteines 122 and 123 to Alanine) quantitatively reduces its attachment to mucin granules. Expression of KChIP3 WT in HT29-18N2 cell lines stably depleted of KChIP3 inhibits mucin secretion, whereas expression of non palmitoylated KChIP3 (KChIP3 AA) only partially rescues the effect of KChIP3 depletion and the cells maintain higher levels of baseline secretion compared to KChIP3-WT cells. Altogether, our data suggest that zDHHC3 or zDHHC4 dependent palmitoylation is involved in KChIP3 recruitment to mucin granules to control the baseline mucin secretion.

Lipoprotein N-Acylation in Staphylococcus aureus Is Catalyzed by a Two-Component Acyl Transferase System

ABSTRACT Bacterial lipoproteins (Lpps) are a class of membrane-associated proteins universally distributed among all bacteria. A characteristic N-terminal cysteine residue that is variably acylated anchors C-terminal globular domains to the extracellular surface, where they serve numerous roles, including in the capture and transport of essential nutrients. Lpps are also ligands for the Toll-like receptor 2 (TLR2) family, a key component of the innate immune system tasked with bacterial recognition. While Lpp function is conserved in all prokaryotes, structural heterogeneity in the N-terminal acylation state is widespread among Firmicutes and can differ between otherwise closely related species. In this study, we identify a novel two-gene system that directs the synthesis of N-acylated Lpps in the commensal and opportunistic pathogen subset of staphylococci. The two genes, which we have named the lipoprotein N-acylation transferase system (Lns), bear no resemblance to previously characterized N-terminal Lpp tailoring enzymes. LnsA (SAOUHSC_00822) is an NlpC/P60 superfamily enzyme, whereas LnsB (SAOHSC_02761) has remote homology to the CAAX protease and bacteriocin-processing enzyme (CPBP) family. Both LnsA and LnsB are together necessary and alone sufficient for N-acylation in Staphylococcus aureus and convert the Lpp chemotype from diacyl to triacyl when heterologously expressed in Listeria monocytogenes. Acquisition of lnsAB decreases TLR2-mediated detection of S. aureus by nearly 10-fold and shifts the activated TLR2 complex from TLR2/6 to TLR2/1. LnsAB thus has a dual role in attenuating TLR2 signaling in addition to a broader role in bacterial cell envelope physiology. IMPORTANCE Although it has long been known that S. aureus forms triacylated Lpps, a lack of homologs to known N-acylation genes found in Gram-negative bacteria has until now precluded identification of the genes responsible for this Lpp modification. Here, we demonstrate N-terminal Lpp acylation and chemotype conversion to the tri-acylated state is directed by a unique acyl transferase system encoded by two noncontiguous staphylococci genes (lnsAB). Since triacylated Lpps stimulate TLR2 more weakly than their diacylated counterparts, Lpp N-acylation is an important TLR2 immunoevasion factor for determining tolerance or nontolerance in niches such as in the skin microbiota. The discovery of the LnsAB system expands the known diversity of Lpp biosynthesis pathways and acyl transfer biochemistry in bacteria, advances our understanding of Lpp structural heterogeneity, and helps differentiate commensal and noncommensal microbiota.

Characterization and expression of Cm-AAT1 gene encoding alcohol acyl-transferase in melon fruit (Cucumis melo L.) ‘Hikapel’

Wibowo WA, Fatkhurohman MI, Daryono BS. 2020. Characterization and expression of Cm-AAT1 gene encoding alcohol acyl-transferase in melon fruit (Cucumis melo L.) ‘Hikapel’. Biodiversitas 21: 3041-3046. Melon (Cucumis melo L.) is one of the horticulture commodities that have high economic value and its needs increase continuously. Many new melon cultivars have been assembled to produce a higher quality melon. Melon 'Hikapel' developed by the Laboratory of Genetics and Breeding, Faculty of Biology UGM has distinctive character in the form of a strong aroma. This aroma is a complex mixture of various kinds of volatile compound. One of the main determinant compounds is a volatile ester, synthesized by the alcohol acyl-transferase enzyme encoded by the Cm-AAT1 gene. Characterization of Cm-AAT1 began with isolation of melon rinds to get total RNAs. Synthesis cDNA was conducted with oligo-dT primer, followed by detection of Cm-AAT1 using specific primers. A specific band was sequenced to perform phylogenetic tree. Gene expression from 4 melon cultivars, ‘Hikapel’, ‘Hikadi’, ‘Sun Lady’, and ‘Luna’ analysis was performed using relative quantitative Real-Time PCR. The results of this study showed that Cm-AAT1 owned not only by aromatic cultivars ‘Hikapel’ and ‘Hikadi’, but also owned by non-aromatic cultivars ‘Sun Lady’ and ‘Luna’. Phylogenetic analysis shows a high similarity between Cm-AAT1 on 'Hikapel' and 'Hikadi'. Gene expression analysis on 'Hikapel' increases as the process of fruit ripening during the storage period and it is in contrast to 'Hikadi' at decrease when the fruit began to enter the decay process on day 7th. Expression of Cm-AAT1 on ‘Hikapel’ was higher than ‘Hikadi’ at the peak of fruit maturity.